Mastering Cryopreservation for Maximum Cell Recovery

1 How To Guide Cells and tissues are valuable, sometimes irreplaceable assets in life sciences, making the ability to store cells and tissue samples safely an essential consideration in laboratory life. Long-term storage of samples requires a method that prevents contamination, preserves function and minimizes genetic change. This allows consistency and reliability of results across research projects and sustains established cell lines that may be expensive or time consuming to replace. Cryopreservation – the cooling of living tissue or cells to extremely low temperatures – can achieve all of these things, when performed successfully. Low temperatures can significantly decrease or even stop the biological and chemical reactions of cells, preserving them in stasis without killing them, allowing for further research or application when thawed. However, simple freezing or storage at incorrect temperatures can damage and destroy cells due to the formation of ice crystals and increased solute concentrations. Therefore, specific protocols using cryopreservative agents (CPAs) are required to protect the cells and tissues.1 The concept of cryopreservation as we know it today was first developed in the 1940s, with the discovery that glycerol could be used as a CPA.2 While preserving cells is now a routine technique in most laboratories, current research suggests that whole organs could be cryopreserved, potentially transforming organ donation and allowing a biobank of on-demand transplants.3 This guide will discuss the common methods for cryopreservation in the lab, explore current methods and challenges and provide tips and tricks to assist you in selecting the most appropriate techniques and reagents to help optimize your long-term cell storage. Selecting the right materials and method The basic method for cryopreservation is simple. Healthy, viable cells are counted and harvested (either through enzymatic or mechanical means), then centrifuged and resuspended in appropriate freezing media for the cell type. The cell suspension is then aliquoted into cryovials and stored at -80 °C overnight, before transition to liquid nitrogen (LN) for long term storage. However, the viability and stability of cryopreserved cells can be easily affected by inefficient protocols. There are a wide range of aspects to consider when cryopreserving cells and optimizing your techniques can help ensure maximum recovery rates and reduce variation between lots. Healthy cells The most important aspect to consider for successful cryopreservation is the cells themselves. After all, even the best reagents and equipment available can’t ensure viable recovered cells if the samples that were frozen down were subpar or contaminated. Mastering Cryopreservation for Maximum Cell Recovery Kate Harrison, PhD MASTERING CRYOPRESERVATION FOR MAXIMUM CELL RECOVERY 2 How To Guide Best practices for harvesting and freezing cells includes proper aseptic technique – working inside a laminar flow hood or biosafety cabinet and using a 70% alcohol spray to maintain sterile conditions. Cells should also be tested for contamination – particularly mycoplasma contamination – before harvesting. For the best recovery of viable cells, cells should be harvested at a low passage number, during their log growth phase. Extended passages can result in genetic changes over time, potentially causing genotypic or phenotypic alterations and affecting research. If creating a master stock for future experiments, early passage numbers are therefore essential for freezing, before the cell line has a chance to change. Cells are ready to harvest at 70–80% confluency, when they are still actively growing and before they reach stationary phase. The optimum concentration of cells for cryopreservation varies between cell lines but is often within the range of 1 x 106–5 x 106 cells/mL. Freezing cells outside the optimum range, at too high or too low a density can impact viability and recovery rates. Freezing density should be kept as consistent as possible to reduce variability. Cryogenic vials Cells can’t be frozen down in just any old vials or snap-cap tubes. Cryovials are specially designed to withstand the extremely low temperatures of LN, and to remain durable over long periods. Most cryovials are now made from polypropylene, which is cheaper, lighter and shatter-resistant, compared to the more traditional glass. Cryovials come in different sizes and styles, and it’s important to use the best type of vial for your needs. Size should be considered, with most vials able to store volumes of 1 mL to 5 mL. It is important not to overfill vials, allowing space for samples to expand into as they freeze, preventing vial rupture – e.g., a 1.2 mL vial should be selected for sample sizes of up to 1 mL. Choosing vials with graduated markings can also help prevent overfilling. When selecting vial size, it’s also important to consider how many aliquots you will need. Aliquots should be used over one large stock, to avoid repeated freeze–thaw cycles. For samples that will be stored in LN, screw capped cryovials are essential to prevent accidental opening during extreme temperature changes. But when it comes to screw-cap tubes, even the threading can make a difference. Internally threaded vials take up less storage space, but externally threaded vials can reduce contamination risk. Finally, ensure you choose a vial that offers sufficient space on the surface for a barcode or written information – for long-term storage, detailed records and tracking are essential, otherwise staff changes could render samples useless. Some new vials have radio frequency identification tags compatible with ultra-cold temperatures, while future cryovials may benefit from LN resistant chips able to store batch information and storage history.4 Cryoprotective agents and freezing media Freezing media is typically made up of the normal media for your particular cell culture, fetal bovine serum (or another source of protein) and a CPA. CPAs are a key consideration during cryopreservation, as they prevent cells from damage during freezing by decreasing the freezing point and reducing both the amount of ice formed and the salt concentration in the solution.4 CPAs fall into two categories: permeating agents and non-permeating agents. A good permeating CPA should be highly water soluble at low temperatures, good at crossing biological membranes and show low levels of toxicity. Some common permeating CPAs include glycerol, dimethyl sulfoxide (DMSO), ethylene glycol and propylene glycol (PG).5 Their small size allows them to enter cells, where they bind intracellular water MASTERING CRYOPRESERVATION FOR MAXIMUM CELL RECOVERY 3 How To Guide molecules, decreasing the freezing level and preventing ice crystal formation by blocking the interaction of water molecules with each other. Instead, CPA binding causes the water molecules to become solid without the formation of ice – a process known as vitrification.6 Non-permeating CPAs include polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), raffinose, sucrose and trehalose. These agents also induce vitrification and prevent high concentrations of salts, but in an extracellular manner. All CPAs have some level of toxicity, and so must be added in a careful, stepwise manner at appropriate concentrations. In addition, different cryoprotectants are suited to different cell types or situations. For example, DMSO is the most common CPA, with relatively low toxicity to cells and effective ice protection. However, it can cause DNA methylation and other epigenetic changes, so is not best placed for cryopreservation of embryos and oocytes.7 In comparison, PG is less toxic than DMSO so better for preserving more sensitive cells, but has lower cryoprotective efficacy, so needs to be used at higher concentrations. When preparing to freeze down a new cell line or type of tissue sample, careful literature research and trial freezing experiments should be performed to optimize the type and concentration of CPA used and maximize recovery of healthy samples. Freezing process In addition to ice crystal formation, the exposure of cells and tissues to cold temperatures can cause damage, in the form of cold shock and chilling injury. Chilling injury is the damage caused by holding cells at temperatures far below their normal functional temperature for too long, while cold shock is the damage caused by sudden large decreases in temperature. Therefore, once the CPA/cell solution is made and decanted into cryogenic vials, the cooling rate – not too fast, not too slow – must be carefully managed to avoid either type of damage. An appropriate cooling rate must allow sufficient time for water to leave the cell and vitrification to occur. For the majority of cells, maximum sample survival is achieved with a rate of approximately 1 °C per minute.8 The exception to this is very large cells, such as hepatocytes, which require greater membrane permeability or slower cooling rates.5 The most accurate, reproducible and documentable method of achieving this freezing rate is with a programed controlled-rate freezer. However, these can be expensive, difficult to use and require a large footprint, and so are not found in most labs. Instead, cells can be placed in either an isopropanol-containing freezing container, or an alcohol-free polyethylene foam container, and then inside a -80 °C freezer overnight. Cells should then be moved as soon as possible to an LN tank for long-term storage at a temperature below -135 °C. If necessary, cells can be stored at -80 °C for up to a month, but this should be avoided wherever possible to support sample viability and stability. Cells can be stored in LN for years and remain stable and viable, however it is recommended that cells are stored in the vapor phase of LN rather than the liquid phase to prevent contaminations and reduce the chance of cryovial explosion when thawing.9 LN must always be handled with appropriate training, ventilation and safety measures in place. Thawing If preserving viability demands a slow freeze, the opposite is true of thawing. Current understanding suggests that cells should be thawed as rapidly as possible – often in a 37 °C water bath – in order to prevent ice recrystallization.10 This should be approached with caution though, as water baths are often sources of contamination and should be cleaned regularly. The potential toxicity of CPAs also warrants rapid thawing followed by prompt dilution in culture medium to minimize any toxic effects. This should be followed by counting the cells and assessing viability (e.g., with trypan blue) before experimental use. Once an aliquot of cells is thawed, it should not be refrozen. Repeated freeze–thaw cycles should be avoided as this can decrease cell viability, damage protein structure and affect DNA integrity. MASTERING CRYOPRESERVATION FOR MAXIMUM CELL RECOVERY 4 How To Guide Key takeaways Cryopreservation of cells may appear simple, but it’s still important to make sure you have an optimized method, or your cells could suffer! Healthy cells from the outset are essential, so make sure your initial cultures are viable, confluent and contamination free. You may need to do some optimization experiments and extra research to find the right cryovial and CPA for your particular cells or tissues, but this extra time will pay dividends when you have a stable, reproducible stock of cells to work from in the long term. References 1. Pegg DE. The relevance of ice crystal formation for the cryopreservation of tissues and organs. Cryobiology. 2010;60(3):S36-S44. doi:10.1016/j.cryobiol.2010.02.003 2. Sztein JM, Takeo T, Nakagata N. History of cryobiology, with special emphasis in evolution of mouse sperm cryopreservation. Cryobiology. 2018;82:57-63. doi:10.1016/j.cryobiol.2018.04.008 3. Han Z, Rao JS, Gangwar L, et al. Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model. Nat Commun. 2023;14(1). doi:10.1038/s41467-023-38824-8 4. Hunt CJ. Technical considerations in the freezing, low-temperature storage and thawing of stem cells for cellular therapies. Transfus Med Hemoth. 2019;46(3):134-150. doi:10.1159/000497289 5. Whaley D, Damyar K, Witek RP, Mendoza A, Alexander M, Lakey JR. Cryopreservation: An overview of principles and cell-specific considerations. Cell Transplant. 2021;30. doi:10.1177/0963689721999617 6. Fahy GM, Wowk B. Principles of cryopreservation by vitrification. Method Mol Bio. Published online November 14, 2014:21-82. doi:10.1007/978-1-4939-2193-5_2 7. Verheijen M, Lienhard M, Schrooders Y, et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci Rep. 2019;9(1). doi:10.1038/s41598-019-40660-0 8. Mazur P. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol. 1963;47(2):347-369. doi:10.1085/jgp.47.2.347 9. Tedder RS, Zuckerman MA, Brink NS, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet. 1995;346(8968):137-140. doi:10.1016/s0140-6736(95)91207-x 10. Baboo J, Kilbride P, Delahaye M, et al. The impact of varying cooling and thawing rates on the quality of cryopreserved human peripheral blood T cells. Sci Rep. 2019;9(1). doi:10.1038/s41598-019-39957-x About the Author Kate has a BSc in Microbiology from the University of Manchester and a PhD in virology from the University of Edinburgh. She now works at Technology Networks as a senior science writer, where she is responsible for the creation of custom written content.